Antithrombin III, or heparin cofactor, is the major plasma inhibitor of the coagulation proteases. Alone, antithrombin III reacts slowly with these enzymes to form a 1:1 inactive complex. Addition of heparin, however, greatly enhances the rate of protease inactivation without altering the stoichiometry of the reaction. This increase in reactivity appears to result from heparin induced conformational changes in antithrombin III and the coagulation proteases. We propose to prepare specific chromophoric derivatives of antithrombin III to further define the heparin induced conformational changes. Also, we will characterize the interaction of antithrombin III with a series of serine proteases including thrombin, factor Xa, factor IXa, and trypsin. The chromophoric reporter groups will be used to measure alterations in antithrombin III structure resulting from the formation of the individual inhibitor-protease complexes. The conformational changes will be correlated with the kinetics of protease inactivation. These approaches should enable us to identify conformational features of antithrombin III that determine its relative reactivity towards the individual proteases.